EP1699289A1 - Non-human mammal comprising a modified serca2 gene and methods, cells, genes, and vectors thereof - Google Patents
Non-human mammal comprising a modified serca2 gene and methods, cells, genes, and vectors thereofInfo
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- EP1699289A1 EP1699289A1 EP04808891A EP04808891A EP1699289A1 EP 1699289 A1 EP1699289 A1 EP 1699289A1 EP 04808891 A EP04808891 A EP 04808891A EP 04808891 A EP04808891 A EP 04808891A EP 1699289 A1 EP1699289 A1 EP 1699289A1
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- gene
- serca2
- cell
- serca
- rodent
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Definitions
- the present invention relates to a non-human vertebrate, particularly a non-human mammal, whose germ-cells and somatic cells comprise a modified sarco(endo)plasmic reticulum Ca 2+ ATPase (Serca ATPase) gene. Further, the present invention relates to a method of inducing defective Ca handling, as well as a method for studying heart failure in non-human vertebrates, particularly non-human mammals. The invention also relates to a modified Serca ATPase gene, as well as eukaryotic cells and vectors containing this gene.
- One purpose of the invention is to provide a tool to genetically affect Ca 2+ handling in live animals in an organ of interest; e.g. in heart muscle to produce heart failure.
- the invention is particularly useful for studying heart failure and as a tool for developing novel drugs and methods for treatment of heart failure.
- Heart failure is defined as a pathophysiological state in which the heart is unable to pump blood to meet the body's metabolic demand. Heart failure has a high morbidity and mortality (2-5 years) after diagnosis. The number of heart failure patients has doubled the last 12 years in Western society, and results in a heavy load on the healthcare system. The most common causes are high blood pressure, repeated episodes of ischemia or myocardial infarction. Especially for the latter patient group, one expects an increase in heart failure at a later stage because of the increase in acute infarction survival rate.
- the three major determinants of contractile function in cardiac muscle cells are the trigger of Ca 2+ release, reuptake of Ca 2+ into the sarcoplasmic reticulum (SR) by Serca ATPases, and available SR Ca 2+ content.
- SR sarcoplasmic reticulum
- the hallmark functional changes in post-infarction heart failure are an increase in the muscle relaxation time after contraction, reduced muscle fractional shortening, and reduced contractile force in the cardiomyocytes (Holt, T ⁇ nnessen et al. 1998; Hasenfuss and Pieske 2002).
- Serca ATPases are major ATP-driven Ca 2+ handling ion pumps in the cell.
- Ca 2+ handling refers herein to the transportation of Ca 2+ inside the cell, in and out of organelles, and in and out of the cell.
- Serca proteins exist as several protein isoforms, coded by the three genes Sercal, Serca2 and Serca3 (Loukianov, Ji et al. 1998; Wuytack, Raeymaekers et al. 2002, and references therein) Sercal is predominant in fast-twitch skeletal muscle, Serca3 is expressed mainly in secretory tissues and in organs such as kidney and pancreas, whereas Serca2 is expressed in all tissues in the body.
- the Serca2a isoform is expressed in cardiomyocytes and slow-twitch skeletal muscle (very low levels in other tissues). In cardiac and slow-twitch skeletal muscle, Serca2 plays an essential role in muscle contraction.
- the Serca2b isoform is expressed in most cell types, including vascular smooth muscle, CNS, and non-neuronal tissue. Recently, a new isoform expressed in monocytes, Serca2c, was described in humans (Gelebart, Martin et al. 2003). Muscle pathology has been associated with mutations in the Serca class genes in humans (Loukianov, Ji et al. 1998; Shull, Okunade et al. 2003).
- Sercal and Serca2 genes in mice are neoneatal and early embryonic lethal, respectively (Periasamy, Reed et al. 1999; Pan, Zvaritch et al. 2003). In contrast, Serca3 null mice are viable, but display other defects (Arredouani, Guiot et al. 2002).
- Serca2 activity, Serca2 mRNA and protein content in failing hearts of both humans and experimental animals are reduced compared with non-failing hearts. It has been clearly demonstrated that increasing Serca2 expression level, either by adenoviral infection or in transgenic mice, improved both contractility and SR function in experimental animals (Periasamy and Huke 2001; Nakayama, Otsu et al. 2003). It has also been demonstrated that Sercal is able to partially substitute for Serca2 function in the heart (Loukianov, Ji et al. 1998).
- amino acid sequence similarity between the Sercal, 2 and 3 protein isoforms within one species is very high (>98%), as exemplified by the alignment of murine Serca proteins isoforms shown in figure 1.
- Furthennore, all the Serca proteins are highly conserved in mammals. This high level of conservation is exemplified by the amino acid alignment of Serca2 from a range of mammalian species provided in figure 2.
- Serca2 t/nu11 heterozygous mice While a null mutation in the Serca2 gene (atp2a2) in mice is embryonic lethal, Serca2 t/nu11 heterozygous mice have a reduction in Serca2 protein and decreased contractility in the heart (Periasamy, Reed et al. 1999; Ji, Lalli et al. 2000; Huke, Liu et al. 2003). h a Serca2 isoform-specific mutant mouse model, Serca 2b b (Serca2a nu11 ) animals develop cardiac hypertrophy (Ver Heyen, Heymans et al. 2001). Both models have a 30-40% reduction in Serca2 ATPase protein and activity. Nevertheless, none of these mice develop heart failure.
- the present invention overcomes the above obstacles by providing a transgene non- human animal, in which the level of Serca ATPase function, more particular Serca2 ATPase, can be directly manipulated in adult animals in order to simulate defect Ca 2+ - handling in general, and heart failure in particular.
- This effect is obtained by employing inducible recombination systems, like for example Cre-loxP.
- inducible recombination systems like for example Cre-loxP.
- Such systems allow both spatial (a given cell type) and temporal (a given time) control of mutations in living animals.
- the invention also comprises and methods, cells, vectors and gene sequences thereof.
- the system of Cre-loxP animals is based on two separate animal lines carrying two genetic components (described in U.S patent number 4,959,317).
- One animal line carries the gene of interest marked by loxP recombination sites (floxed gene). These sites are placed such that the gene of interest is inactivated when recombination takes place between the two recombination sites. This specific recombination reaction is catalyzed by the Cre recombinase.
- the second animal line expresses the Cre recombinase and directs the timing of gene destruction in the tissue or organ of interest. When these two animal lines are, for example, bred together, the gene of interest is excised in the tissue or organ of interest.
- a similar recombination system for mammals has been developed, based on the fit recombination site and FLP recombinase for PI bacteriophage.
- the present invention is a valuable tool for studying medical conditions caused by defect Ca 2+ handling, especially heart failure, as well as screening for, and further testing of, substances antagonizing such conditions.
- MLC-2v wt/cre IVSd/s, inter- ventricular septum thickness in diastole respectively systole; LVDd/s, left ventricular diameter in diastole respectively systole; FS, fractional shortening in LVD; PWd/s, posterior wall thickness in diastole respectively systole; LAD, left atrial diameter; PWSV, posterior wall shortening velocity; PWRV, posterior wall relaxation velocity; LVOT, left ventricular outlet tract; RVOT, right ventricular outlet tract; CO, cardiac output; VTI, velocity time integral. All values are mean ⁇ SEM
- Fig. 1 Sequence alignment of mouse Serca 1, 2 and 3 protein. Amino acid identity is designated by a dot. Gaps are designated by -. Amino acid substitutions in the Serca family proteins relative to mouse Sercal protein are shown. Accession numbers for mouse Serca ATPases: mouse la, NP_031530; mouse 2a and 2b, NP_033852 and J131870; Serca3a, AAB04099; Serca3b, AAB04098; Serca3c, CAA75746.
- Fig. 2 Sequence similarity of Serca2 proteins in mammalian species. Amino acid identity is designated by a dot. Gaps are designated by -. Amino acid substitutions in the Serca2 proteins in other species relative to mouse Serca2 are shown. Accession numbers for Serca2 ATPases: mouse 2a and 2b, NP_033852 and J131870; rat 2a, P11507; rat 2b, NP_058986; human 2a, NP_001672; human 2b, NP_733765; human 2c, AAO47398; dog 2a, 046674; cat 2a, Q00779; pig 2a and 2b, PI 1607; rabbit 2a and 2b, P20647.
- Fig. 3 Targeting construct for Serca2 flox gene modification. Sequence information. Partial DNA sequence information of the targeting vector is given. Gaps in the sequence are given by lines and text of positioning in the atp2a2 gene. Introns are in italics. Coding sequence is in capital letters. Extra inserted material (cloning sites, antibiotics cassettes and loxP sites) is underlined. LoxP sites are in bold. Elements are placed into atp2a2 gene intron 1, and intron 3 (2 positions). See figure 2 for schematic. The Serca2 gene sequence can be obtained from GenBank accession: NM_009722.
- Fig. 4 A) Schematic representation of genetic manipulation. A simple representation of the genetic elements introduced into the Serca2 gene introduced into the animal invention. B) Verification of Serca locus targeting events offspring from chimeric mice. Offspring from chimeric animals were genotyped by PCR. The intron 1 loxP site and intron 3 loxP sites were detected by primer pairs OL88/85 and OL86/87, respectively. DNA from Balb/C and from ES cell clone #1 were included as wild type and Serca2 flox allele controls. Fractions indicate dilutions of tail DNA preparations
- Fig. 5 Specificity of gene deletion in a test model. Detection of Serca2 alleles in mouse tissues.
- Fig. 6 Cardiac ANP mRNA expression.
- the expression level of ANP was estimated by Northern analysis. Blot were sequentially probed with [32P]-dCTP-labelled probe for ANP.
- Fig. 7 Serca2 protein expression.
- Total protein homogenates of the indicated tissues from control and cardiac heterozygous Serca2 mice were run on 8% SDS-PAGE gels and analysed by Western blotting. Appropriate antibodies are given in the materials and methods section.
- Total lysate/lane heart, 15 ⁇ g, other tissues 40 ⁇ g.
- VV is heart lysate as positive control.
- Genotypes: (Control) FF Serca2 flox/flox (Cardiac heterozygous)
- FC Serca2 wt flox MLC-2v wt/cre -
- Serca2a isoform in heart and soleus muscle
- Serca2b isoform in heart, soleus and EDL muscle
- Fig. 8 Compensatory mechanisms in Serca flox MLC-2v-Cre mice.
- Total protein homogenates from hearts from control and cardiac heterozygous Serca2 mice were run on SDS-PAGE gels (7% for NCXl, 15% for phospolamban) and analysed by Western blotting.
- Gel loading was lO ⁇ g protein sample except for detection of phosphoSerl ⁇ PLB (50 ⁇ g/sample) and NCXl in soleus + EDL (40 ⁇ g). Samples were denatured at 95 °C for 5-10 min (A, C) or at 37 °C for 15 min (B) to avoid complete disruption of PLB pentamers.
- Genotypes: (Control) FF Serca2 flox/flox (Cardiac heterozygous)
- FC Serca2 wt/fl0X MLC-2v wt/cre
- Panel A Total phospholamban, phospho-Serl ⁇ PLB, and phospho-Serl ⁇ PLB, monomeric
- Panel B Total phospholamban, phospho-Serl ⁇ PLB and phospho-Serl7 PLB, pentameric Panel C) NCXl .
- the band present in EDL muscle is not NCXl .
- Genotypes PCR Generation of animals with Serca2 flox and MCM transgene alleles.
- FIG. 10 Heart morphology. Whole hearts from FF (ID 8.3 and 7.4) and KO (LD 8.1 and 7.3) animals were removed at day 43 or day 52 days after induction of gene excision with tamoxifen. Note the strongly enlarged left atrium in the KO animal (7.3) after 52 days.
- Fig. 11 Pilot series left atrial diastolic diameter Tamoxif en-injected mice were examined by echocardiography at the indicated time points. The left atrial diastolic dimension was the most sensitive parameter of heart dilatation. All KO animals had dilated left atria compared with FF controls
- Fig. 12 Serca protein content in tamoxifen-induced FF and KO mice.
- Roman numerals denote pilot experiments I, II and III.
- FF and KO denote genotypes Serca2 flox/flox and Serca2 flox/flox MCM respectively. Days after tamoxifen injection are noted at the top.
- Sercal and NCXl B Western blot with 60 ⁇ g total protein for all 6 KO animals and 10 ⁇ g total protein for 2 FF control animals.
- the present invention provides a non-human vertebrate all of whose cells comprise a modified Serca ATPase gene with inserted recombination sites.
- Said Serca ATPase gene modification may be homozygous or heterozygous; present as one or several copies, with or without the co-presence of a wild-type Serca ATPase gene.
- the Serca ATPase gene may be integrated in the genome of the host or exist in an extra- chromosomal vector. Any Serca ATPase type or isoform can be employed in view of the high level of sequence similarity and conservation of function of the Serca family (See figure fig. 1), but preferably the ATPase gene is a Serca2 ATPase gene.
- Recombination sites can be inserted at any position adjacent or inside the Serca gene to cause complete or partial destruction of the gene when combined with a suitable recombinase.
- any recombination sites suitable for eukaryotic cells can be used to carry out the present invention, like, for example, ftr or loxP recombination sites.
- the recombination sites are of heterogenous origin, more preferably of non-mammalian origin, and most preferably the recombination sites are loxP recombination sites.
- the recombination sites can be inserted in one or both alleles of the gene to cause a partial or complete destruction of Serca gene product activity.
- the introduced loxP sites are placed such that both isoforms of the Serca2 genes (Serca2a and Serca2b) are disrupted in the tissue of interest when excision occurs.
- Serca2a and Serca2b is expressed in the heart and in slow-twitch skeletal muscle, whereas the longer Serca2b protein is expressed in all tissues examined to date.
- the vertebrate, more preferably the mammal, of the cunent invention can further comprise a recombinase of non-mammalian origin.
- This recombinase must be able to interact with the recombination sites of the modified Serca ATPase gene and effect inactivation of the gene.
- the recombinase can be of any type (e.g. FLP or Cre recombinase) provided it does not affect endogenous recombination sites, but preferably, the recombinase is a Cre recombinase.
- the recombinase gene can be expressed in all cells of the vertebrate or only in certain tissues like, for example, tissue specific expression in skin, muscle, brain, kidney, or subgroups of these.
- a recombinase gene is expressed in the heart ventricle and in slow-twitch skeletal muscle.
- the recombinase gene can be exclusively, predominately, or modestly expressed in the tissue of interest compared to other tissues.
- the differential expression can vary greatly, but it is prefened that the recombinase is expressed exclusively or predominately in the tissue at stake.
- the recombinase gene can be expressed by induction, i.e. controlled by a regulatory nucleic acid sequence, preferably an inducible promoter or regulatory nucleic acid sequence that controls the expression of the gene, or extra fused gene material which controls the intracellular Cre recombinase location in the cell.
- An endogenous or exogenous inducing agent can effect the activation of this inducible promoter or regulatory sequence.
- the former are the tet system promoter, while examples of the latter are fusion of mutant estrogen receptor domain to the recombinase which confers tamoxifen inducibility.
- Methods to introduce a recombinase gene and its promoter or regulatory sequence into a eukaryotic cell are within the knowledge of a person skilled in the art.
- the non-human mammal may be any mammal except a human being.
- the mammal is a non-human mammal generally used as animal models in pharmaceutical and scientific research and development, e.g. porcine, canine, or rodent animal models, more preferably a rodent, and most preferably a mouse.
- Genes homologous or analogous to Serca ATPases exist in all vertebrates, and vertebrate model systems other than non-human mammals are consequently possible, e.g. fish, more particularly Zebra fish (Branchydanio reri ⁇ ).
- Another aspect on the present invention is a eukaryotic cell comprising a modified Serca ATPase gene with inserted recombination sites.
- the Serca ATPase gene, recombination sites, and regulatory nucleic acid sequences are as described supra.
- the cell is preferably of vertebrate or mammalian origin, more preferably of non-human mammalian origin, even more preferably of rodent origin, and most preferably of mouse origin.
- the cell may be of any particular type, for example, a cardiomyocyte, a keratinocyte, a neuron, an embryonic cell, etc. Most preferably, the cell is an embryonic cell.
- the cell may further comprise a non-native or heterogenous recombinase gene, more preferably a non-mammalian recombinase gene, such as a FLP or Cre recombinase gene.
- a non-mammalian recombinase gene such as a FLP or Cre recombinase gene.
- the recombinase gene is a Cre recombinase gene.
- Yet another aspect of the present invention is a modified Serca ATPase, or a related vertebrate gene, with inserted recombination sites.
- the Serca ATPase gene, recombination sites, and regulatory nucleic acid sequences are as described supra.
- Suitable recombination sites can be inserted at any position adjacent or inside the Serca gene to cause complete or partial inactivation of the gene when present in a host cell. Any types of recombination sites can be used provided endogenous recombinases do not bind and effect recombination at the site when present in a host cell.
- the recombination sites are of heterogenous origin, more preferably of non-mammalian origin.
- the recombination sites are loxP recombination sites.
- the ATPase gene which is to be modified, is preferably a Serca2 ATPase, and most preferably as listed in Genbank SEQ ID NM_009722. According to a most prefened embodiment, said Serca ATPase is modified substantially as listed in SEQ ID 1 ( Figure 3). Here, a modification of the flanks of the Serca2 gene, as employed in a most prefened embodiment, is depicted.
- the gene sequence of the Serca gene can be degenerated as a result of the redundancy of the genetic code. Also, codons can be changed with codons encoding similar amino acids.
- Another aspect of the present invention is a vector comprising the above Serca ATPase gene.
- the vector may be of any type appropriate for the task, for example, a shuttle vector, vectors for replication in eukaryotes or prokaryotes only, yeast vectors, etc.
- the vector is based on pBluescript II KS.
- the present invention also provides a (1) method for inducing defective Ca 2+ handling in a non-human vertebrate, preferably a non-human mammal, and a (2) method for inducing heart failure in a non-human vertebrate, preferably a non-human mammal.
- the first method comprises the steps of inducing recombination and inactivation of a Serca ATPase gene in a non-human vertebrate as described supra.
- Introduction of the gene can be done, for example, at the embryonic level by embryo fusion or by gene-therapeutic methods like exposing a live animal to a modified adenovirus carrying a suitable recombinase. In the latter case, the virus can then be targeted to specific tissues of the animal.
- the non-human vertebrate animals are, as earlier described, preferably standard laboratory animals, like e.g. fish, preferably Zebra fish, monkeys, pigs, dogs, sheep, goat, guinea pigs, or rodents, more preferably rodents, and most preferably mice.
- the Serca ATPase gene is preferably of type 2 and the recombination sites can be inserted at any position adjacent or inside the Serca gene to cause complete or partial destruction of the gene in combination with a suitable recombinase.
- the recombination sites are of heterogenous origin, more preferably of non-mammalian origin. Most preferably, the recombination sites are loxP recombination sites.
- the recombination sites can be inserted in one or both alleles of the gene to bring about partial or complete destruction of Serca gene product activity. Methods to introduce recombination sites into eukaryotic cells are known in the art.
- the recombinase gene is preferably of heterogenous origin, more preferably of non-mammalian origin, and most prefened a Cre recombinase.
- the recombinase is preferably controlled by an inducible promoter sequence or another inducible regulatory sequence. It is prefened that the recombination gene is only, or predominantly, expressed in specific tissues, as described above. This can be any tissue, but most preferably heart tissue.
- the second method comprises the steps of inducing recombination and inactivation of a Serca ATPase gene in heart tissue.
- any type of method can be used to produce animals with heart tissue comprising both a modified Serca gene and a nonnative or heterogenous recombinase. These methods include for example gene therapy, embryo fusion, etc.
- the step of mating or gene introduction can of course be omitted in cases where suitable model animals already have been produced.
- the non-human vertebrate is preferably a standard laboratory animal, like exemplified in the method above.
- the recombinase gene is preferably controlled by an inducible promoter sequence or another inducible regulatory sequence, for example a tamoxifen-inducible promoter as described supra.
- an inducible promoter sequence or another inducible regulatory sequence for example a tamoxifen-inducible promoter as described supra.
- an inducible promoter sequence or another inducible regulatory sequence for example a tamoxifen-inducible promoter as described supra.
- a tamoxifen-inducible promoter as described supra.
- Ca 2+ handling in heart tissue is partially or fully disturbed by the tissue specific induction of the recombinase.
- the reduced Ca 2+ pumping ability depending on the level of reduction, will lead to effects varying from decreased heart contractility to massive heart failure.
- a rodent all of whose cells comprise a Serca2 gene modified with loxP sites, is mated with a rodent partner, all of whose cells comprise a Cre recombinase, to produce progeny with cells comprising both said Serca2 gene and Cre recombinase gene.
- a mouse all of whose cells comprise a Cre recombinase, but which is predominantly or only expressed in the heart ventricle and slow-twitch skeletal muscle; is mated with another mouse whose cells comprise a Serca2 ATPase gene modified with loxP recombination sites.
- Yet another aspect of the present invention is a method for screening a compound or a mixture of compounds for activity against defective Ca 2+ handling, comprising the steps of inducing recombination and inactivation of a Serca ATPase gene in a non-human vertebrate, preferably a non-human mammal; administrating the compound or mixture to said vertebrate before and/or after the induced inactivation of the Serca ATPase gene; and observe for effects.
- the Serca ATPase gene and non-human vertebrate are as described above and the Serca ATPase gene is inactivated by means of expressing a recombinase, also as described above. Parameters observed might, for example, be improvement in general condition, alleviation of symptoms, reduced lethality, and improvement of heart contractility.
- Another aspect of the invention is a method for screening a compound or a mixture of compounds for activity against heart failure, comprising the steps of inducing recombination and inactivation of a Serca ATPase gene in heart tissue of a non-human vertebrate; administrating the compound or mixture to said mammal before and or after the induced inactivation of the Serca ATPase gene; and observe for effects.
- the Serca ATPase gene and non-human vertebrate are as described above and the Serca ATPase gene is inactivated by means of expressing a recombinase, also as described above. Parameters observed might, for example, be improvement in general condition, alleviation of symptoms, reduced lethality, and improvement of heart contractility.
- introducing a gene into a vertebrate or "introducing a gene into a vertebrate” are herein intended to mean any method for producing animals whose cells comprise both a recombinase and a modified Serca gene, both as described above. Examples of such methods are mating, gene therapy and embryo fusion.
- the non-human animal all of whose cells comprise a modified Serca ATPase gene can be bred with animal lines canying the Cre recombinase expressed in the tissue of interest.
- available animal Cre lines may direct Serca2 gene inactivation specifically in the heart in adult animals, by induction with tamoxifen (Sohal, Nghiem et al. 2001). This particular combination would provide a genetic means of inducing heart failure in a standardized experimental animal model. This model should be applicable for large-scale therapeutic drug screening and development.
- the major disadvantage of traditional animal models where heart failure is induced by operative procedures is the difficulty of standardizing operative procedures and their effects on the animals, and the difficulty of large-scale experiments required for therapeutic drug development.
- Serca2 gene inactivation in live mice is detennined by the properties of the Cre recombinase-expressing mouse to which it is mated.
- mice expressing the Cre recombinase specifically in the heart, brain, liver, lymphoid system or keratinocytes have been made, and can be bred to Serca2 ox mice to inactivate the Serca2 gene in those particular organs or tissues of interest.
- the major application for Serca2 flox mice would be to generate mouse models for human diseases/disorders involving the Serca2 gene by breeding with appropriate recombinase-expressing mice.
- the mouse model reflects the phenotype of the human disorder
- the mouse model should be a valuable test model for drug development.
- Inactivation of the Serca2 gene in the heart may induce heart failure, while inactivation of the Serca2 gene in keratinocytes may induce dermatological conditions.
- inactivation of the Serca2 gene in the brain may induce neurological dysfunction and inactivation of the Serca2 gene in the immune cells may induce immunological dysfunction.
- CHF congestive heart failure
- Serca2 In humans, mutations in the Serca2 gene have been linked with the skin disorders keratosis follicularis (Dariers's disease) and acrokeratosis verruciformis (Dhitavat,
- DD Darier's disease
- keratosis follicularis in humans is an autosomal dominant skin disorder characterized by loss of adhesion in epidermal cells and abnormal keratinization (Cooper and Burge 2003 & Hovnanian 2004).
- Cunent treatment is rudimentary.
- the Serca2 mutations found in Dariers 'patients result in reduced Serca2 function, Ca 2+ sensitivity or protein expression level in a heterologous expression assay in HEK-293 cells (Dode et al, 2003). However, there is no formal proof that the identified mutations result in loss of function and the observed phenotypes.
- a mouse model to test for the loss of Serca2 gene function in skin would therefore be necessary to prove the link between genotype and phenotype.
- Our invention can be bred such that the Serca2 gene is inactivated only in keratinocytes. Such an animal would be the only available animal model for these diseases and for drug development.
- a null mutation in the Serca2 gene (atp2al) in mice is embryonic lethal, and demonstrates the necessity of this protein (Periasamy, Reed et al. 1999). Mutations in the human atp2a2 gene have been linked to dermatological disorders (Cooper and Burge 2003; Dhitavat, Macfarlane et al. 2003), and it is a matter of debate whether some of these mutations also are linked to neuropsychiatric disorders (Jacobsen, Lyons et al. 1999; Jacobsen, Franks et al. 2001; Wang, Yang et al. 2002).
- Animal husbandry and breeeding Animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals (NLH Publication No. 85-23, revisedl996). Mice were housed in M2 cages with Bee Kay bedding (Scanbur BK) in 55% humidity on a 12 h light/dark cycle. Food pellets (mouse + rat standard ex., Scanbur BR) and water was freely available.
- Echocardiography and hemodynamic pressure measurements were performed as described (Finsen 2004). In short, mice were anaestetized, intubated and connected to a rodent ventilator using 2% isoflurane. Two-dimentional, M-mode and Doppler echocardiography was performed using a il3L 13MHz linear anay transducer (GE) designed for the examination of small rodents. Data were analyzed with EchoPac PC software (GE). Hemodynamic measurements were performed sequentially with a 1.4 F Millar micro-tipped pressure transducer catheter (SPR-671, Millar Instruments, Houston, TX) advanced into the left ventricle.
- GE linear anay transducer
- Ca 2+ ATPase content Total Ca 2+ ATPase in mouse heart left ventricles was measured by an ATP-binding assay in tissue homogenates as described elsewhere (Everts, Andersen et al. 1989; Holt and Christensen 1997).
- mice embryonic stem cell line ES14.1a (stock at the KTTC, Karolinska Institute, Sweden) was used for genetic manipulations.
- Serca2 flox mice All genetic manipulations were performed using standard molecular biology techniques.
- the Serca2 (atp2a2) gene was cloned from a commercial genomic mouse lambda phage library (129 SVJ from Stratagene).
- the targeting vector was named pSerca2T. All manipulations were confirmed by DNA sequencing (see figure 3).
- the targeting vector was introduced into mouse embryonic stem cells (ES cells) by standard techniques (electroporation).
- ES cells canying homologous recombination events with conectly placed loxP sequences were enriched by antibiotics selection procedures, and identified by Southern hybridization techniques and PCR.
- the antibiotics selection genes were then excised by a second round of electroporation with a Cre recombinase-expressing plasmid.
- Conect clones were again identified by Southern blotting and PCR.
- the final result of the genetic manipulations was that two loxP sites and additional necessary cloning and restriction sites were introduced into 2 separate introns in the atp2a2 gene. The modification is outlined in figure 4.
- Conectly genetically modified ES cells canying 2 loxP sites in one allele of the atp2a2 gene were then used to generate genetically modified mice (Serca2 flox mice) canying these loxP recombination sites.
- the loxP sites are placed such that the atp2a2 gene is converted into a null allele when the Cre recombinase is appropriately expressed in the organ or tissue of interest. Subsequent production of all isoforms of Serca2 Ca 2+ ATPase protein is reduced or eliminated in the tissue of interest
- mice The mouse line was backcrossed onto the B6/J background (N10), and bred to homozygosity.
- Serca2 wt, flox or deletion Different alleles of the Serca2 gene (Serca2 wt, flox or deletion) and the MLC-2v gene (wt and cre knock-in) were detected by standard PCR reactions with annealing temperature of 55 °C for 25-30 cycles and Amplitaq Gold on an ABI 9600 thermocycler (Applied Biosystems).
- Primer combinations and PCR amplification product sizes are given below: Position Primer pair PCR product Serca2 intron 1 OL84 / OL85 wt: 247 bp flox: 419 bp Serca2 intron 1 OL88 / OL85 flox: 318 bp Serca2 intron 3 OL86 / OL87 wt: 282 bp flox: 381 bp Serca2 deletion OL88 / OL87 324 bp Cre (knockin) OL102 / OL103 340 bp MLC-2v wt OL104 / OL105 183 bp
- Tissue material hnmediately after conclusion of the in vivo measurements, the mice were sacrificed with excision of heart, lung, liver, kidney, spleen, soleus and extensor digitorum longus (EDL) muscles. Hearts and lungs were blotted dry and immediately weighed. All organs were flash frozen in liquid nitrogen.
- Protein content was measured by a bicinchoninic acid assay (Pierce 23235) using BSA as standard protein.
- Samples were electrophoresed in SDS-PAGE gels (8% or 15%) and transferred to 0.45 ⁇ m PVDF membranes by standard procedures.
- Membranes were blocked in 5% skim milk in TBS-T (A ershani Life Sciences) or in an alternative BPPT blocking solution (5% BSA, 1% polyvinylpynolidone-10, 1% polyethyleneglycol, MW 3500, 0.2% Tween 20 in 2xTBS) (NanoTools Antik ⁇ rpertechnik, info@nanotools.de) where indicated.
- the following antibodies were used for protein detection: monoclonal antibodies against Serca2a (MA3-919) , phospholamban PLB (MA3-922) (all Affinity Bioreagents), polyclonal anti-Serca2b antiserum(Campbell, Wuytack et al. 1993) (kind gift from Frank Wuytack), anti-NCXl (Thomas, Sjaastad et al. 2003), anti-phospho-Serl6-PLB and anti-phospho-Thrl7-PLB (both Badrilla).
- Serca2 flox mice were intercrossed with MLC-2v Cre knock-in mice (Chen, Kubalak et al. 1998; Minamisawa, Gu et al. 1999) in which the Cre recombinase is expressed in the heart ventricle and in slow-twitch skeletal muscle with no detectable basal expression in other tissues.
- the Serca2 flox and MLC-2v Cre knock-in mice were at generation N3 and N3 onto the B6/J background before interbreeding. In the final breeding between
- Serca2 flox/flox and Serca2 wt flox MLC2v wt cre animals we were unable to obtain offspring with the expected homozygous "cardiac knockout" genotype (Serca2 flox/flox MLC2v t/cre ) (table 1). More than 100 offspring were genotyped. In the six mating pairs (with alternation of parental genotypes), we could detect offspring with only 2 of the 4 expected genotypes (Serca2 flox/flox MLC2v wt/wt and Serca2 wt/flox MLC2v wt cre ).
- mice Animal physiology The animal age, heart, lung and body weights were measured in Serca2 t/flox MLC- 2v wt/cre double heterozygous mice and in control littermates (Serca2 wt/flox MLC-2v wt wt ) (Table 2). In total, hearts and organs were examined from 71 animals. There were no gross anatomical differences. Overall, there was no significant difference between the two genotypes in animal size, heart and lung weight, or in the ratios of heart weight body weight or lung weight/body, indicative of no overt disease in the double heterozygous animals. As expected, females had significantly lower body weight, heart weight and lung weight than males. However, no significant differences were detected between the genotypes within each sex.
- the amount of functional Ca 2+ ATPase enzyme in hearts from both genotypes was measured by an ATP-binding assay (table 5).
- Serca2a and Serca2b isofomis were examined in both left ventricle and in slow-twitch soleus muscle by Western blotting with isoform-specific antibodies.
- Serca2a was reduced by 28% and 26% in double heterozygous Serca2 wt/flox MLC-2v /cre mice (figure 7, panel A) compared with control Serca2 flox/flox MLC-2v wt/ t littermates.
- a parallel decrease in cardiac Serca2b protein was also detected, 49% in double heterozygotes compared to control animals (panel b).
- Serca2a and Serca2b protein was reduced by 25% and 35% in the double heterozygotes compared to control animals (figure 7, panel B).
- other tissues such as lung, spleen, liver and kidney, there was no decrease in Serca2b expression in the double heterozygous animals relative to controls (figure 7, panel B, lower part).
- Serca2a and Serca2b proteins were reduced specifically in the "cardiac Serca2 heterozygous" animals in the heart and in slow-twitch soleus muscle, but not in other tissues.
- Atrial natriuretic factor is readily induced in stressed, hypertrophic or dysfunctional hearts. No differences in ANP expression between the two genotypes was detected, arguing against the hearts being in a stressed state at a subclinical level (figure 6).
- NCXl RNA expression was increased by 40%> (figure 8, panel C). In EDL and soleus muscle, there was no detectable NCXl immunoreactivity. This is in keeping with data from the Serca2 heterozygous mouse (Periasamy, Reed et al. 1999), and provides an explanation for compensatory pathway for removing Ca 2+ from the cytosol out of the cell via NCXl, since the expression level of Serca2, and removal of Ca 2+ into the SR, is reduced
- Serca2 flox mice of the invention with MLC-2v Cre mice.
- Serca2a was partially inactivated in the heart and in slow skeletal muscle of Serca2 wt/flox MLC-2v wt/cre double heterozygous mice as shown by PCR, ATPase and protein measurements.
- the invention can be used to generate standardized animal models with 74- defective Ca handling in the tissue of organ of interest, and thus be useful in biomedical research and therapeutic drug development.
- Serca2 flox mice were the bred to transgenic MerCreMer mice, which express the Cre recombinase in the heart under control of the alpha-myosin heavy chain promoter (Sohal, Nghiem et al. 2001).
- the presence of Cre recombinase in the nucleus, and thereby activity, is controlled by tamoxifen.
- Cardiac Serca2 knockout mice will be produced by injection of Serca2 flox flox MerCreMer with tamoxifen as described (Sohal, Nghiem et al. 2001). The animals will be characterized as the heterozygous animals of Examples 1 to 2. In addition, the forward and reverse mode functions of Serca2 ATPase in cardiac SR vesicles will be measured.
- One predicted phenotype of this cardiac restricted Serca2 knockout mouse can be full compensation because other proteins are able to sufficiently handle the Ca 2+ movements in the cardiomyocyte (e.g. NCXl). The other extreme phenotype may be sudden death due to a collapse in the electrochemical balance in the cardiomyocytes (anhythmic phenotype).
- the animals are evaluated by ECG to detect anhythmic activity.
- ECG ECG
- the heart will first have sufficient time to develop compensatory mechanisms, but then progress into a failing state as the Serca2 function is severely reduced.
- the heart may display a hypertrophic phenotype with activation of cytokine systems and other signal transduction pathways. This model would then mimic the compensated and failing stages of heart failure in humans. Given the latter scenario, the endpoint will be death. This is a "hard" endpoint ideal for the invention's proposed use as a tool for developing improved drugs and therapies against heart failure.
- another feasible feature will be the extended time window which will permit time for conducting the experiment as well as monitoring.
- Serca2 flox mice were the bred to transgenic MerCreMer (MCM) mice, which express the Cre recombinase in the heart under control of the -myosin heavy chain promoter (Sohal, Nghiem et al. 2001).
- MCM transgenic MerCreMer
- Cre recombinase is fused to two copies of a mutant estrogen receptor ligand binding domain which bind the estrogen antagonists tamoxifen and 4-OH tamoxifen.
- the expressed transgene is localized in the cell cytoplasm in cardiac myocytes. Upon binding to tamoxifen, the transgene enters the nucleus and permit excision of the Serca2 flox allele. Thus, excision of the Serca2 flox allele is controlled both by tissue specificy and the presence of tamoxifen.
- Serca2 flox and transgenic MCM mice generated the expected genotypes at the expected frequencies (table 6 and figure 9).
- Serca2 flox/flox MCM mice breed normally, and show no overt signs of cardiac dysfunction or other abnormal physiology.
- Tamoxifen (1 mg/lOO ⁇ l in peanut oil or sunflower seed oil) was administered i.p. to each animal at dosages of 1 mg/day for 5 days or for 2x5 days (weekdays).
- mice In the first pilot series, 4 mice, two of each genotype: Serca2 flox/f!ox (denoted FF) and Serca2 flox flox MCM (denoted KO) received tamoxifen i.p. at 1 mg/day for 5 days. After 43 days one FF and one KO animal were examined and no overt signs of heart failure were seen. Hearts were removed for biochemical analysis. After 57 days the remaining KO animal died of severe heart failure with a severely enlarged left atrium, enlarged right ventricle, increased lung weight, fluid in the thorax and abdomen, whereas the FF mouse was completely normal. In the second pilot series using 4 animals, two of each genotype as above received tamoxifen i.p.
- mice displayed clinical signs of heart failure such as sluggishness, fluid in the thorax and abdomen at the most advanced stage, tachypnoe and poor general condition.
- the animals displayed dilated left atrium and right ventricle.
- left ventricular function was also compromised. There were no indications of hypertrophy in the myocardium.
- This model should be suitable as a general heart failure model without the interference of hypertrophic processes.
- the animals also may be useful as a model for right-sided cardiac dysfunction.
- SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility.
- Serca2 Flox heterozygous control Serca2 wt/flox MLC-2v wt wt 0 0 25 0
- Serca2 Flox homozygous control Serca2 flox/flox MLC-2v wl/wt 44 44 25 50
- Cardiac Serca2 heterozygous Serca2 wt flox MLC-2v wt/cre 56 55 25 50
- Body and organ weights for Serca2 cardiac heterozygous and control mice Age, body, heart and lung weights of control and heterozygous animals (Mean ⁇ SEM).
- Body weight (g) 33.4 ⁇ 1.7 10 32.9 ⁇ 2.0 9
- FC Serca2 wt flox MLC-2v wt cre
Abstract
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